The long-term objective is an understanding of the mechanism of protein-mediated reactions on cell surfaces by using newly developed pre-steady-state transient kinetic techniques. The proteins are the neurotransmitter receptors at the junction between about 10(12) cells of the mammalian nervous system and the transport proteins that remove the neurotransmitters from these junctions, thus terminating the reactions. The specific aims are to determine the reaction mechanisms of these proteins. What are the rate and equilibrium constants of the reactions and how are they affected by disease-causing mutations of the proteins, therapeutically useful compounds, and abused drugs? The principal investigator will investigate the excitatory nicotinic acetylcholine and glutamate receptors, the inhibitory gamma-aminobutyric acid (GABA) receptor, and the dopamine transporter. Among the compounds that inhibit or activate these proteins are the anti-convulsant MK-801, the abused drug cocaine, neurosteroids, and combinatorially synthesized RNA ligands that inhibit or prevent inhibition. The principal investigator will investigate the mechanism of a GABA receptor reported to function abnormally in the hippocampus of epilepsy-prone rats. Once one understands the mechanisms one can explore the selection of compounds that counteract receptor malfunction. Health Relatedness of the Project: Neurotransmitter receptors regulate intercellular communication in the central nervous system and provide the mechanism by which environmental information is received, stored, and transmitted. They are implicated in nervous system diseases (e.g., epilepsy, Parkinson's) and are the targets of therapeutic agents (for instance anti-convulsants, tranquilizers) and abused drugs. An understanding of their effects on the protein-mediated reaction mechanisms can lead to the rational clinical treatment of diseases and the design of improved drugs. Research Design: The principal investigator will use both the steady-state single-channel current-recording technique and the pre-steady-state kinetic techniques newly developed by this laboratory. The new transient kinetic techniques for use with membrane-bound proteins are suitable for studying reactions on cell surfaces in the us-ms time region. Receptors are equilibrated with a photolabile, biologically inert neurotransmitter precursor before photolysis releases the neurotransmitter in us, thereby initiating the reaction. The resulting whole-cell current due to opening of receptor-channels or dopamine uptake is recorded and analyzed. Using combinatorially synthesized RNA ligands for specific sites on the membrane-bound proteins, the principal investigator will test predictions arising from the mechanism based on the kinetic measurements. The ligands are isolated by a technique developed by the principal investigator's laboratory.